Biosynthesis of Tracheal Mucous Glycoproteins

粘液分泌过多是阻塞性肺病的标志，包括慢性支气管炎、哮喘和呼吸道疾病。 囊性纤维化这种情况是粘液细胞肥大和增生的结果。分泌 上皮细胞表面的杯状细胞和粘膜下腺体的粘液细胞，粘蛋白不仅是 粘液分泌物的粘弹性的主要决定因素，但也可以作为受体， 病原体粘蛋白的功能主要存在于碳水化合物中，其占总糖的70-90%。 气道粘蛋白。此外，粘蛋白碳水化合物是非常不均匀的，这使得它们能够 捕获许多不同的空气传播的病原体，并促进它们从呼吸道中清除。粘蛋白碳水化合 核心2、核心4和I型血分支可以扩展结构及其功能潜力 结构.所有这三种结构都可以通过粘液组织特异性核心2 N-乙酰葡糖胺转移酶- M（C2GnT-M）。C2 GnT-M基因表达的调节可以极大地影响理化性质 气道粘蛋白的性质和气道粘液的功能。C2 GnT-M基因的表达可被抑制 EGF的作用，而视黄酸和Th 2细胞因子的作用增强。C2 GnT-M活性也可以在 衬底水平。C2 GnT-M的缺失在结直肠癌中已有报道，其重新表达可以抑制结直肠癌的发生。 结肠癌细胞的致瘤性。因此，C2 GnT-M的改变不仅可以对细胞增殖产生显著影响， 健康，还有疾病。本申请的目的是表征C2 GnT-M的调节 在酶活性和基因表达的水平上。我们建议：1.确定的活性位点 通过X射线晶体学和定点诱变，然后测量酶， 使用核心1、核心3和血型I二糖受体及其同系物的活性。2. 通过定位顺式调控元件和鉴定转录，表征C2 GnT-M基因调控 在基础和Th 2干扰素处理条件下的因子。这些转录因子将被鉴定， 用已知转录因子的cDNA转染并用生物素化的启动子下拉， 用转录因子蛋白阵列测定。将通过EMSA和CHIP试验对其进行表征。3. 确定C2 GnT-M是否可以作为气道上皮粘液细胞的特异性标志物。目前的研究可以 导致通过鉴定小的 碳水化合物抑制剂、Th 2型丝氨酸诱导的转录因子和粘液细胞特异性启动子。

Mucus hypersecretion is a hallmark of obstructive lung diseases, including chronic bronchitis, asthma, and cystic fibrosis. This condition is the result of hypertrophy and hyperplasia of mucus cells. Secreted from goblet cells on the surface epithelium and mucus cells in the submucosal glands, mucins not only are the major determinant of the viscoelastic properties of mucus secretion but also can serve as the receptors for pathogens. The functions of mucins reside primarily in the carbohydrates, which constitute 70-90% of airway mucins by weight. In addition, mucin carbohydrates are very heterogeneous, which allow them to trap many different airborne pathogens and facilitate their removal from the airways. Mucin carbohydrate structures and their functional potential can be expanded by core 2, core 4, and blood group I branch structures. All three structures can be formed by mucus tissue-specific core 2 N-acetylglucosaminyltransferase- M (C2GnT-M). Modulation of C2GnT-M gene expression can greatly affect the physicochemical properties of airway mucins and functions of airway mucus. Expression of C2GnT-M gene can be inhibited by EGF but enhanced by retinoic acid and Th2 cytokines. C2GnT-M activity also can be regulated at the substrate level. Loss of C2GnT-M has been reported in colorectal cancer and its re-expression can inhibit tumorigenicity of colonic cancer cells. Thus, alteration of C2GnT-M can have a significant impact on not only health but also diseases. The objective of this application is to characterize the modulation of C2GnT-M at the levels of enzyme activity and gene expression. We propose to: 1. Determine the active site of C2GnT-M by X-ray crystallography and site-directed mutagenesis followed by measurement of enzyme activities using core 1, core 3, and blood group i disaccharide acceptors and their homologues. 2. Characterize C2GnT-M gene regulation by mapping cis-regulatory elements and identifying the transcription factors under basal and Th2 cytokine-treated conditions. These transcription factors will be identified by transfection with cDNAs of known transcription factors and pull-down with biotinylated promoter followed by assay with transcription factor protein array. They will be characterized by EMSA and CHIP assay. 3. Determine if C2GnT-M can be a specific marker for airway epithelial mucus cells. Current studies could lead to the development of therapy for mucus hypersecretory diseases through identification of small carbohydrate inhibitors, Th2 cytokine-induced transcription factors, and mucus cell-specific promoter.